JP2018100906A - Method for separating chiral amino acids - Google Patents

Abstract

An object of the present invention is to provide a simple, precise and high-throughput method for separating and analyzing chiral amino acids, chiral dipeptides and chiral tripeptides in a sample.
A method for separating one or more of a chiral amino acid, a chiral dipeptide and a chiral tripeptide in a sample using liquid chromatography, wherein the amino group is AQC derivatized and then weakly anion-exchanged. A method for separating using a liquid chromatography in which a first chiral column having a stationary phase and a second chiral column having an amphoteric ion exchange type stationary phase are connected.
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Description

  The present invention relates to a method for separating chiral amino acids, chiral dipeptides or chiral tripeptides.

  Amino acids are one of the main group of compounds that make up life forms. In amino acids other than glycine, the content ratio of amino acid enantiomers in vivo is large and biased toward the L-form, and it has long been thought that in higher animals such as mammals, only L-amino acids exist and have physiological functions. Has been. However, in recent years, it has become clear that D-amino acids are also present in the body of mammals with the progress of analytical methods, and it is becoming clear that they have physiological functions.

  Regarding the separation and analysis of chiral amino acids, both molecular species separation between mixed amino acids and L-type and D-type chiral separation in each molecular species are required. Conventionally, for example, an amino acid is converted to an NBD derivative using a 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) reagent, and then a reverse phase column (first dimension: molecular species separation). ) And a chiral column having a stationary phase carrying a chiral identifier (second dimension: chiral separation) using a two-dimensional liquid chromatography (LC) method (Non-patent Document 1) or a single reversed-phase column ( One-dimensional LC method using ODS column (Non-patent Document 2), Amino acid 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized, One-dimensional LC method using one chiral column (Non-patent document 2) Patent Document 3) has been devised.

However, although the two-dimensional LC method performs separation of molecular species and chiral separation under separation conditions suitable for each, the separation performance is excellent, but the apparatus and operation are complicated because a flow path switching valve and a multi-loop are required. In addition, when a multi-component target is used, a sufficient separation time difference is required between target components in the first dimension, and thus low throughput is cited as a problem.
On the other hand, the one-dimensional LC method, which has been reported in recent years and can be carried out simply, is difficult to sufficiently satisfy the separation of both the molecular species and the chiral species, and the analytical accuracy caused by the overlap between amino acids or contaminating components. Is a problem point.

Hamase K, Miyoshi Y, Ueno K, Han H, Hirano J, Morikawa A, Mita M, Kaneko T, Lindner W, Zaitsu K., J Chromatogr A. 2010 Feb 12; 1217 (7): 1056-62. Doi: 10.1016 / j.chroma.2009.09.002. Epub 2009 Sep 6. Mochizuki T, Takayama T, Todoroki K, Inoue K, Min JZ, Toyo'oka T., Anal Chim Acta. 2015 May 22; 875: 73-82.doi: 10.1016 / j.aca.2015.02.054. Epub 2015 Feb twenty three. Karakawa S, Shimbo K, Yamada N, Mizukoshi T, Miyano H, Mita M, Lindner W, Hamase K., J Pharm Biomed Anal. 2015 Nov 10; 115: 123-9.doi: 10.1016 / j.jpba.2015.05. 024.Epub 2015 Jun 16.

  The present invention relates to providing a method capable of separating all molecular species of chiral amino acids in a sample easily, with high accuracy and with high throughput.

  As a result of studying a method for separating chiral amino acids, chiral dipeptides and chiral tripeptides using liquid chromatography, the present invention has achieved chromatographic separation by combining two ion exchange chiral columns having stationary phases carrying different chiral identifiers. It has been found that chiral amino acids, chiral dipeptides and chiral tripeptides can be separated accurately and simply.

That is, the present invention is a method for separating any one or more of a chiral amino acid, a chiral dipeptide and a chiral tripeptide in a sample using liquid chromatography, wherein the amino group is AQC derivatized and then weakly anion exchanged. The present invention relates to a method for separation using liquid chromatography in which a first chiral column having a stationary phase of a type and a second chiral column having a zwitterion exchange type stationary phase are connected.
In addition, the present invention includes the step of identifying and quantifying any one or more of a chiral amino acid, chiral dipeptide and chiral tripeptide by mass spectrometry following the above method, and separating and quantifying chiral amino acid, chiral dipeptide or chiral tripeptide A method is provided.

  According to the method of the present invention, it is possible to separate chiral amino acids in a sample, for example, all chiral amino acids constituting a protein, chiral dipeptides and chiral tripeptides with high accuracy, simply and with high throughput. Therefore, simultaneous analysis of chiral amino acids becomes possible, which can be an extremely useful tool for chiral amino acid metabolome analysis.

Results of chiral amino acid analysis of standard products. Results of chiral amino acid analysis of stratum corneum sample. Quantitative value of chiral amino acid in stratum corneum sample. Results of chiral amino acid analysis by chiral column reverse connection. Results of chiral amino acid analysis using each chiral column alone. Results of analysis of chiral dipeptide standards. Analysis results of chiral tripeptide standard.

  The method of the present invention is a method for separating any one or more of a chiral amino acid, a chiral dipeptide and a chiral tripeptide in a sample using liquid chromatography, wherein the amino group is AQC derivatized and then weakly anion exchanged. The separation is performed using liquid chromatography in which a first chiral column having a stationary phase of a type and a second chiral column having a zwitterion exchange type stationary phase are connected.

  In the present invention, examples of chiral amino acids include 20 types of amino acids constituting proteins, and D-type (D-amino acids) and L-types (L-amino acids) of metabolism-related amino acids (for example, ornithine and citrulline). 20 types of amino acids (D-type and L-type) are preferred. The chiral dipeptide or chiral tripeptide is an oligopeptide consisting of 2 or 3 molecules of the chiral amino acid. Hereinafter, chiral amino acids, chiral dipeptides and chiral tripeptides are collectively described as “chiral amino acids” or simply “amino acids”.

In the present invention, the sample containing protein or amino acid is, for example, a part of a human, an animal, a plant, etc. In the case of a human or an animal, skin, skin stratum corneum, hair, organ, blood collected from a living body or a dead body , Body fluids, and cells and tissues reconstituted therefrom.
For example, the skin-derived sample is collected non-invasively by tape stripping or the like.
The collected sample is immersed in a solvent such as water or an organic solvent (methanol, ethanol, 2-propanol, chloroform, tetrahydrofuran, etc.), and amino acids and peptides are extracted by a conventional method. Furthermore, when there is a possibility that the chiral amino acid analysis is affected by the contaminating component, a pretreatment for removing the contaminating component may be performed.
Peptides and proteins consisting of 4 or more amino acids can be derivatized and separated according to the present invention after hydrolysis into amino acids according to a known method. Therefore, it is possible to analyze chiral amino acids constituting peptides and proteins.

In the method of the present invention, chiral amino acids can be separated as long as at least the chiral amino acid separation means is performed by liquid chromatography (LC). The method for identifying and quantifying the separated chiral amino acid is not particularly limited.
Chiral amino acid identification and quantification methods include UV-visible detector, diode array detector, fluorescence detector, differential refractive index detector, evaporative light scattering detector, charged particle detector, mass spectrometer, etc. -Although the method of quantifying is mentioned, it is preferable to use a mass spectrometer (MS) from a viewpoint of highly sensitive and highly selective detection. Therefore, the separation and quantification method including the method of the present invention is more preferably performed using LC-MS, LC-MS / MS, or the like that combines liquid chromatography (LC) and mass spectrometry (MS).

In the present invention, when the above sample is applied to liquid chromatography, the amino acid is converted to an AQC derivative in which the amino group is carbamoylated using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The
The method of derivatizing AQC can be performed according to a known method. For example, the amino acid standard solution or amino acid sample, borate buffer solution, and AQC solution are mixed, immediately stirred and then heated.
Thus, samples containing AQC-derivatized amino acids are prepared without particular dilution.

In the present invention, separation of chiral amino acids by liquid chromatography is performed by placing an amino acid sample derivatized with AQC in an apparatus including two different types of chiral columns for chromatography.
Here, as the chiral column to be used, a chiral column having two different chiral identifiers having an ion exchange type chiral stationary phase (CSP) is used.
The first chiral column has a weak anion exchange type (WAX) stationary phase, for example, a substrate in which O-9- (tert-butylcarbamoyl) quinine is held as a chiral identifier on a substrate, or O-9 -(Tert-Butylcarbamoyl) quinidine retained (refer to A below).
Such chiral columns are commercially available as “CHIRALPAK QN-AX” and “CHIRALPAK QD-AX” (both DAICEL).
On the other hand, the second chiral column has an amphoteric ion exchange type stationary phase. For example, quinine (8S) in which (S, S) -trans-2-aminocyclohexanesulfonic acid is bound as a chiral identifier to a base material. 9R) or (R, R) -trans-2-aminocyclohexanesulfonic acid-bound quinidine (8R, 9S) is retained (see B below).
Chiral columns having such a stationary phase are commercially available as “CHIRALPAK ZWIX (+)” and “CHIRALPAK ZWIX (−)” (both DAICEL).

  Examples of the base material (support) that holds such a chiral identifier (indicated by “●” in the above formula) include a porous organic base material or a porous inorganic base material, preferably a porous inorganic base material. . Suitable materials for the porous organic substrate are high molecular weight materials made of polystyrene, polyacrylamide, polyacrylate, etc., and suitable materials for the porous inorganic substrate are silica gel, alumina, magnesia, glass, kaolin, titanium oxide. Silicate, hydroxyapatite, and the like. A particularly preferred substrate is silica gel.

  When silica gel is used as the substrate, the particle size of the silica gel is 1.7 μm to 10 μm, preferably 3 μm to 5 μm, and the average pore size is 50 to 150 μm, preferably 100 to 150 μm.

  The column can also have suitable dimensions. For example, the inner diameter may be 2.1 to 20 mm and the length may be 10 to 250 mm, preferably the inner diameter is 2.1 to 4.6 mm and the length is 150 to 250 mm.

  In the present invention, the connection mode of the first chiral column and the second chiral column is not limited, but is preferably connected in series from the viewpoint of simultaneous separation of chiral amino acids. In the case of series connection, the order is not particularly limited.

In the method of the present invention, liquid chromatography can select various separation modes. That is, either a gradient mode or an isocratic mode can be selected. The isocratic mode is preferable.
When selecting an isocratic mode, for example, it is desirable to use a high polarity mobile phase. Preferred is an excellent proton-donating solvent methanol type, and examples thereof include a methanol-water mixed solution and a methanol single solution.

  In any separation mode, if necessary, the separation and / or detection of derivatized amino acids can be facilitated to improve chromatographic peak shape and / or to promote ionization of LC-MS. Can be formulated with one or more reagents, such as acidic or basic additives. Preferred is a buffer solution system of formic acid and a salt thereof (for example, ammonium formate).

  In the separation and quantification method of chiral amino acids, chiral dipeptides or chiral tripeptides including the method of the present invention, when liquid chromatography and mass spectrometry are used in combination, the mass spectrometer communicates with the liquid chromatography and the amino acids in the sample A mass spectrometry data set can be generated to identify Equipment suitable for performing liquid chromatography-mass spectrometry is commercially available, for example, a Triple Quadrupole LC / MS system manufactured by Agilent Technologies can be used.

  The mass spectrometer includes an ion source for ionizing the separated amino acid molecules to produce charged ion molecules. Examples of ionization methods include electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), electron ionization (EI), fast atom bombardment (FAB) / liquid secondary ionization. Method (LSIMS), matrix-assisted laser desorption ionization method (MALDI), field ionization method, electrolytic desorption method, thermal spray / plasma spray ionization method, particle beam ionization method, etc., among which electrospray ionization method is preferable .

The positively or negatively charged ions produced by ionization are analyzed and the mass to charge ratio (ie m / z) is measured. Suitable analyzers for measuring the mass-to-charge ratio include a quadrupole mass spectrometer (Q-MS), a time-of-flight mass spectrometer (TOF-MS), and an ion trap mass spectrometer (IT-MS). Single type mass spectrometers such as Fourier transform mass spectrometer (FT-MS), hybrid mass spectrometers such as Q-TOF and IT-TOF, or tandem mass spectrometers such as triple quadrupole type (MS However, it is preferable to measure the mass-to-charge ratio using a quadrupole, and it is more preferable to use a triple quadrupole type.
In addition, the detection of product ions generally includes a selected ion monitoring (SIM) method, a full scan method, a selective reaction monitoring (SRM) method, and the like. In the present invention, the SRM method can be suitably used.

The following methods are further disclosed herein as exemplary embodiments of the invention.
<1> A method for separating one or more of a chiral amino acid, a chiral dipeptide and a chiral tripeptide in a sample using liquid chromatography, wherein the amino group is AQC derivatized and then weakly anion exchange-type immobilized A method of separating using a liquid chromatography in which a first chiral column having a phase and a second chiral column having a zwitterion exchange type stationary phase are connected.
<2> The weak anion exchange-type stationary phase of the first chiral column holds O-9- (tert-butylcarbamoyl) quinine or O-9- (tert-butylcarbamoyl) quinidine as a chiral identifier on the substrate. <1>.
<3> The amphoteric ion exchange type stationary phase of the second chiral column holds a quinine in which (S, S) -trans-2-aminocyclohexanesulfonic acid is bound to a base material or (R, R) -The method according to <1> or <2>, wherein quinidine to which trans-2-aminocyclohexanesulfonic acid is bound is held.
<4> The method according to any one of <1> to <3>, wherein the first chiral column and the second chiral column are connected in series.
<5> Following the method according to any one of <1> to <4>, a chiral amino acid or chiral dipeptide comprising a step of identifying and quantifying any one or more of a chiral amino acid, chiral dipeptide and chiral tripeptide by mass spectrometry Alternatively, a method for separating and quantifying chiral tripeptides.
<6> The method according to <5>, wherein the mass spectrometry is performed by MS / MS.
<7> The method according to any one of <1> to <6>, wherein the chiral column base material is silica gel.
<8> The method according to any one of <1> to <7>, wherein the separation mode of liquid chromatography is an isocratic mode.
<9> The method according to <8>, wherein the isocratic mode uses a mobile phase containing an alcohol organic solvent.
<10> The method according to <9>, wherein the mobile phase containing the alcohol organic solvent is a methanol-water mixture or methanol.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

Example 1 Simultaneous separation analysis of chiral amino acids in skin stratum corneum (1) Extraction of skin stratum corneum and extraction of amino acids Film masking tape (Teraoka Seisakusho / 465 # 40, 2.5 cm width x 3 cm length) on the forearm of a healthy male ) Was pressed for 10 seconds to peel off. The skin stratum corneum was sampled by changing to a new tape each time it was peeled off and repeating this peeling operation three times continuously at the same location (collecting area = 7.5 cm ² × 3). When the derivatization treatment was not performed immediately after collection, the sample was stored frozen (−80 ° C.).

(2) Preparation of sample solution and standard product Tape (total 3 sheets) from which the skin stratum corneum was collected was placed in a 5 mL screw tube (trade name: Maruemu / No. 2) in methanol: water (9: 1, v / v). The solution was immersed in 3.0 mL and sonicated for 10 minutes at room temperature to extract amino acids. The extract was transferred to a test tube with a stopper, and then the solvent was distilled off under a nitrogen stream, followed by 0.2 mol / L borate buffer (pH 8.9), AccQ • Tag Ultra derivatization reagent, ie, AQC solution (Waters Manufactured: AQC powder dissolved in acetonitrile at a concentration of 3 mg / mL, that is, 10 mmol / L) was mixed in the order of 80 μL and 20 μL (4: 1), respectively, and immediately stirred and then heated at 55 ° C. for 10 minutes. A solution was prepared.

  Similarly, 0.2 mol / L borate buffer (pH 8.9), each concentration 100 μmol / LD, L-amino acid standard solution (20 amino acids of protein constitution: Ala / alanine, Arg / arginine, Asn / asparagine, Asp / aspartic acid, Cys / cysteine, Gln / glutamine, Glu / glutamic acid, Gly / glycine (D and L are not distinguished), His / histidine, Ile / isoleucine, Leu / leucine, Lys / lysine, Met / methionine, Phe / phenylalanine, Pro / proline, Ser / serine, Thr / threonine, Trp / tryptophan, Tyr / tyrosine, Val / valine, dissolved in 0.2 mol / L borate buffer), AQC solution 70 μL, 10 μL, 20 μL, respectively Mix in the order of (7: 1: 2) Immediately after stirring, an amino acid standard solution was prepared by heating at 55 ° C. for 10 minutes.

(3) LC-MS / MS analysis The solution prepared in (2) was subjected to LC-MS / MS analysis under the following conditions to perform separation detection and quantification of various chiral amino acids.
(apparatus)
LC / 1200 Series (Agilent Technologies), Mass Spectrometer / G6460A Triple Quadrupole (Agilent Technologies)

(Chromatographic separation)
Separation chiral column: CHIRALPAK QN-AX <DAICEL> 4.6 mm ID × 150 mm, particle size 5 μm (first chiral column) and CHIRALPAK ZWIX (+) <DAICEL> 3.0 mm ID × 150 mm, particle size 3 μm (second) (Chiral column) in series in this order (45 ° C)
Eluent: 0.1% (v / v) formic acid and 55 mM ammonium formate, methanol: water (90:10, v / v) solution Elution method: isocratic Mobile phase flow rate: 0.25 mL / min
Injection volume: 5 μL

(Mass spectrometry)
Ionization method: Electrospray ionization method (ESI)
Polarity: Positive ion Fragmentor voltage: 135V
Collision energy: 20V
Capillary voltage: 3500V
Nebulizer pressure: 60 psi
Sheath gas temperature: 250 ° C
Sheath gas flow rate: 12 L / min
Drying gas temperature: 350 ° C
Dry gas flow rate: 13 L / min

(Detection mode)
Proton ion addition molecule ([M + H] ⁺ ) as precursor ion, SRM (selected reaction monitoring) by positive ion mode in which AQC fragment ion (m / z = 171) is set as product ion and AQC fragment ion (m Precursor ion scan detection in positive ion mode with / z = 171)

(4) Data analysis The obtained data was developed into a chromatogram having two axes of retention time and ionic strength. As shown in the chromatograms of FIGS. 1 and 2, AQC-derivatized various chiral amino acids were detected by the analysis method of the present invention.
FIG. 3 shows quantitative values of chiral amino acids in the stratum corneum, and Table 1 shows SRM transitions. Precursor ions were set in Q1 (first stage MS), and product ions were set in Q3 (second stage MS).

Example 2 LC-MS / MS analysis of chiral amino acids (chiral column reverse connection)
(1) Preparation of standard product In the same manner as in Example 1 (2), an amino acid standard solution was prepared.

(2) LC-MS / MS analysis The standard solution prepared in (1) was subjected to LC-MS / MS analysis, and elution order analysis of various chiral amino acids was performed. That is, LC-MS / C was the same as in Example 1 (3) except that CHIRALPAK ZWIX (+) (second chiral column) and CHIRALPAK QN-AX (first chiral column) were connected in series in this order. MS analysis was performed.

(3) Data analysis The obtained data was developed into a chromatogram having two axes of retention time and ionic strength. As shown in the chromatogram of FIG. 4, in the analysis method of the present invention, various chiral amino acids were similarly separated even if the connection order of the first chiral column and the second chiral column was reversed.

Comparative Example 1 Separation and detection of chiral amino acids (CHIRALPAK QN-AX and CHIRALPAK ZWIX (+) used alone)
(1) Preparation of standard product In the same manner as in Example 1 (2), an amino acid standard solution was prepared.

(2) LC analysis The standard solution prepared in (1) was analyzed under the following conditions, and various chiral amino acids were separated and detected.

(apparatus)
LC / 1200 series (Agilent Technologies), DAD / 1290 series (Agilent Technologies)

(Chromatographic separation)
Separation Chiral Column: CHIRALPAK QN-AX <DAICEL> 4.6 mm ID × 150 mm, particle size 5 μm or CHIRALPAK ZWIX (+) <DAICEL> 3.0 mm ID × 150 mm, particle size 3 μm (30 ° C.)
Eluent: 0.1% (v / v) formic acid and 25 mM ammonium formate, methanol: water (98: 2, v / v) solution Elution method: isocratic Mobile phase flow rate: 0.4 mL / min
Injection volume: 5 μL
Detection: UV absorption wavelength 260 nm

(3) Data analysis The obtained data was developed into a chromatogram having two axes of retention time and UV absorption intensity. As shown in the chromatogram of FIG. 5, in the methanol system, Chiralpak QN-AX had high molecular species separation ability, but chiral separation was difficult. On the other hand, CHIRALPAK ZWIX (+) had high chiral resolution, but separation of molecular species was difficult.

Example 3 LC-MS / MS analysis of chiral dipeptide (1) Preparation of standard product 0.2 mol / L borate buffer (pH 8.9), each concentration 100 μmol / L D, L-dipeptide standard solution (DL- Ala-DL-Ala, DL-Ala-Gly, Gly-DL-Ala 0.2 mol / L dissolved in borate buffer) and AQC solution were mixed in the order of 70 μL, 10 μL, and 20 μL (7: 1: 2), respectively. Immediately after stirring, a chiral dipeptide standard solution was prepared by heating at 55 ° C. for 10 minutes.

(2) LC-MS / MS analysis The standard solution prepared in (1) was subjected to LC-MS / MS analysis under the same conditions as in Example 1 (3), and various chiral dipeptides were separated and detected.

(3) Data analysis The obtained data was developed into a chromatogram having two axes of retention time and ionic strength. As shown in the chromatogram of FIG. 6, chiral dipeptides were detected under the same conditions as in the analysis of various AQC-derivatized chiral amino acids by the analysis method of the present invention.
Table 2 shows the SRM transition.

Example 4 LC-MS / MS analysis of chiral tripeptide (1) Preparation of standard 0.2 mol / L borate buffer (pH 8.9), each concentration 100 μmol / LD D, L-tripeptide standard solution ( DL-Ala-Gly-Gly, DL-Leu-Gly-Gly, dissolved in 0.2 mol / L borate buffer) and AQC solution were mixed in the order of 70 μL, 10 μL, and 20 μL (7: 1: 2), respectively. Immediately after stirring, a chiral tripeptide standard solution was prepared by heating at 55 ° C. for 10 minutes.

(2) LC-MS / MS analysis The standard solution prepared in (1) was subjected to LC-MS / MS analysis under the same conditions as in Example 1 (3), and various chiral tripeptides were separated and detected.

(3) Data analysis The obtained data was developed into a chromatogram having two axes of retention time and ionic strength. As shown in the chromatogram of FIG. 7, a chiral tripeptide was detected by the analysis method of the present invention under the same conditions as in the analysis of various AQC-derivatized chiral amino acids.
Table 3 shows the SRM transition.

Claims (9)

  A method for separating one or more of a chiral amino acid, a chiral dipeptide and a chiral tripeptide in a sample using liquid chromatography, wherein the amino group is AQC derivatized and then has a weak anion exchange type stationary phase A method for separation using liquid chromatography in which a first chiral column and a second chiral column having an amphoteric ion exchange type stationary phase are connected.   The weak anion exchange type stationary phase of the first chiral column has O-9- (tert-butylcarbamoyl) quinine or O-9- (tert-butylcarbamoyl) quinidine as a chiral identifier on the base material. The method of claim 1.   The amphoteric ion exchange type stationary phase of the second chiral column is one in which quinine having (S, S) -trans-2-aminocyclohexanesulfonic acid bonded to a substrate is held, or (R, R) -trans- The method according to claim 1 or 2, wherein the quinidine to which 2-aminocyclohexanesulfonic acid is bound is retained.   The method according to any one of claims 1 to 3, wherein the first chiral column and the second chiral column are connected in series.   A method according to any one of claims 1 to 4, comprising the step of identifying and quantifying any one or more of a chiral amino acid, chiral dipeptide and chiral tripeptide by mass spectrometry, chiral amino acid, chiral dipeptide or chiral A method for separating and quantifying tripeptides.   6. The method of claim 5, wherein the mass spectrometry is performed by MS / MS.   The method according to any one of claims 1 to 6, wherein the substrate of the chiral column is silk gel.   The method according to any one of claims 1 to 7, wherein the separation mode of liquid chromatography is an isocratic mode.   The method according to claim 8, wherein the isocratic mode uses a mobile phase of an alcohol-based organic solvent.